In Vitro Mutagenesis: Methods and Protocols

529 Pages·2016·13.007 MB·English

by  Andrew Reeves

Methods in
Molecular Biology 1498
Andrew Reeves Editor
In Vitro
Mutagenesis
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB , UK
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In Vitro Mutagenesis
Methods and Protocols
Edited by
Andrew Reeves
Coskata, Inc., Warrenville, IL, USA
Editor
Andrew Reeves
Coskata, Inc.
Warrenville, IL, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6470-3 ISBN 978-1-4939-6472-7 (eBook)
DOI 10.1007/978-1-4939-6472-7
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Prefa ce
In vitro m utagenesis remains an essential tool for molecular biologists, biochemists, and
metabolic engineers in performing both basic and applied research into gene and protein
function. It is a cross-disciplinary research and development approach that has broad appli-
cations in many important fi elds. With the advent of ever more sophisticated investigative
and analytical methods such as next-generation sequencing , emerging, powerful gene-
editing techniques, and comprehensive data analysis software programs to process large
data sets, the molecular biology fi eld is witnessing a magnitude change in the understand-
ing of gene and protein structural and functional relationships at the organismal level. With
these powerful tools at researchers’ disposal, it is now conceivable to design an in vitro
mutagenesis strategy in a genetically manipulable organism, carry out the mutagenesis
experiments in an effi cient manner, generate, retrieve, and analyze the sequence data and
impacted proteins and phenotypes in weeks or months instead of years, all with far greater
insight into the understanding of the cellular and systems biology of the respective
organism(s) under study. The molecular biologist’s “toolkit” is in a sense now complete for
designing, performing, and analyzing all manner of in vitro mutagenesis experiments with
broad applications in basic research and for any conceivable commercial purpose—be it
drug design, m icrobial cell factories development, gene therapy, or generation of model
organisms, among many other applications.
In this volume on I n Vitro Mutagenesis: Methods and Protocols , an important aim was to
provide the beginner practitioner in the fi eld and the more experienced molecular biolo-
gists, biochemists, and metabolic engineers alike a wide variety of updated, novel approaches
to many powerful classical methods of performing in vitro mutagenesis such as transposon
(Tn) mutagenesis , site-directed and random mutagenesis. Additionally, an important
emphasis was placed on emerging, yet powerful, gene- and genome- editing and b ioinfor-
matics methods now being developed and implemented into experimental reality.
The volume is divided into seven sections: The fi rst two sections describe detailed,
novel methods for gene and genome editing of a broad cross section of the living world
(e.g., mammalian, plant, viral, bacterial, and protistan systems using CRISPR/Cas9 ,
TALEN , and Group II intron technologies). These editing technologies are enabling the
facile genetic manipulation of a wide range of cellular systems in the study of fundamental
metabolic processes. Importantly, they are rigorously being employed for the prospect and
promise of attaining major scientifi c breakthroughs in human physiology and medicine.
The next two sections describe (1) a variety of practical bioinformatics approaches for iden-
tifying mutagenesis targets in silico for the rational design of mutagenesis experiments and
(2) a set of diverse, detailed in vitro Tn mutagenesis protocols for model microorganisms
as well as for use in alternative, previously recalcitrant organisms (e.g., archaea) including
genomic sequencing methods to rapidly and completely identify all the Tn insertion sites in
a mutant library. The last three sections cover a wide variety of novel s ite-directed and ran-
dom mutagenesis approaches aimed at gaining a better understanding of protein-protein
and p rotein-cofactor interactions at the structural and functional levels along with their
concomitant effects on an organism’s cellular metabolism. Provided in these sections are
specialized mutagenic PCR methods (e.g., is- epPCR , epPCR using h eavy water , and single
v
vi Preface
primer PCR, et al.) and mutagenesis and cloning methods (e.g., MUPAC, SliP-SliCE ,
et al.). Several of these chapters also describe state-of-the-art bioanalytical techniques and
methods used by experts in the fi eld to allow a more thorough understanding of how the
specifi c mutation impacted the experimental outcome. The rationale being that the begin-
ner practitioner would be able to view the experimental process from conception and design
to completion through follow-up data analysis.
In keeping with the theme of the Methods in Molecular Biology series, each chapter
contains an extensive Notes section which provides and elaborates on specifi c experimental
details, tips, and tricks and thus it is hoped will allow a more rapid, successful implementa-
tion of the method by practitioners in the fi eld whatever their experience level.
I would like to thank the authors, who are all well-known experts in their respective
fi elds, for their contributions and for allowing me to put together what I believe is a timely,
practical, and comprehensive manual on in vitro mutagenesis methods and protocols that
will be embraced by a broad range of practitioners in the fi elds of molecular biology, bio-
chemistry, biochemical and metabolic engineering , biophysics, among other disciplines.
Moreover, this volume on in vitro mutagenesis was specifi cally compiled with an emphasis
on providing a highly accessible manual for current and future researchers—from the begin-
ner practitioner to the advanced investigator—who routinely perform in vitro mutagenesis
experiments on all types of cells at research institutes, academia, and industrial and govern-
ment laboratories.
Lastly, I would like to thank all my past and current colleagues who have inspired me
to keep learning and to pursue knowledge through hard work, persistence, and investiga-
tive scientifi c endeavors.
Warrenville, IL, USA Andrew Reeves
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
SECTION I GENE AND GENOME-EDITING METHODS PART I
1 D esign and Validation of CRISPR/Cas9 Systems for Targeted
Gene Modification in Induced Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . 3
Ciaran M. Lee , H aibao Zhu , T imothy H. Davis ,
Harshahardhan Deshmukh , and Gang Bao
2 M utagenesis and Genome Engineering of Epstein–Barr Virus
in Cultured Human Cells by CRISPR/Cas9. . . . . . . . . . . . . . . . . . . . . . . . . . 23
Kit-San Yuen , C hi-Ping Chan , K in-Hang Kok , and Dong-Yan Jin
3 U se of CRISPR/Cas Genome Editing Technology
for Targeted Mutagenesis in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3
Rongfang Xu , P engcheng Wei , and Jianbo Yang
4 A ll-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated
Multiplex Genome Engineering in Cultured Cells. . . . . . . . . . . . . . . . . . . . . . 41
Tetsushi Sakuma , T akuya Sakamoto , and Takashi Yamamoto
5 C RISPR/Cas9-Mediated Mutagenesis of Human Pluripotent
Stem Cells in Defined Xeno-Free E8 Medium. . . . . . . . . . . . . . . . . . . . . . . . . 5 7
Chew-Li Soh and Danwei Huangfu
6 D evelopment of CRISPR/Cas9 for Efficient Genome Editing
in Toxoplasma gondii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Bang Shen , K evin Brown , S haojun Long , and L. David Sibley
SECTION II GENE AND GENOME-EDITING METHODS PART II
7 G eneration of Stable Knockout Mammalian Cells by TALEN-Mediated Locus-
Specific Gene Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 07
Barun Mahata and Kaushik Biswas
8 E fficient Generation of Gene-Modified Mice by Haploid
Embryonic Stem Cell-Mediated Semi-cloned Technology. . . . . . . . . . . . . . . . 121
Cuiqing Zhong and Jinsong Li
9 I nsertion of Group II Intron-Based Ribozyme Switches into Homing
Endonuclease Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Tuhin Kumar Guha and Georg Hausner
10 G enerating a Genome Editing Nuclease for Targeted Mutagenesis
in Human Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Zhenyu He and Kehkooi Kee
vii
viii Contents
11 U se of Group II Intron Technology for Targeted Mutagenesis
in Chlamydia trachomatis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 63
Charlotte E. Key and Derek J. Fisher
SECTION III BIOINFORMATICS APPROACHES FOR IDENTIFYING
AND ANALYZING MUTAGENESIS TARGETS
12 I n Silico Approaches to Identify Mutagenesis Targets to Probe
and Alter Protein–Cofactor and Protein–Protein Functional Relationships. . . . 181
Brian A. Dow , E sha Sehanobish , and Victor L. Davidson
13 I n Silico Prediction of Deleteriousness for Nonsynonymous
and Splice-Altering Single Nucleotide Variants in the Human Genome . . . . . . 191
Xueqiu Jian and Xiaoming Liu
14 I n Silico Methods for Analyzing Mutagenesis Targets . . . . . . . . . . . . . . . . . . . 1 99
Troy C. Messina
15 M ethods for Detecting Critical Residues in Proteins . . . . . . . . . . . . . . . . . . . . 2 27
Nurit Haspel and Filip Jagodzinski
16 A Method for Bioinformatic Analysis of Transposon Insertion
Sequencing (INSeq) Results for Identification of Microbial
Fitness Determinants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Nengding Wang and Egon A. Ozer
SECTION IV IN VITRO TRANSPOSON MUTAGENESIS METHODS
IN DIVERSE PROKARYOTIC SYSTEMS
17 A pplication of In Vitro Transposon Mutagenesis to Erythromycin
Strain Improvement in Saccharopolyspora erythraea . . . . . . . . . . . . . . . . . . . . . 2 57
J. Mark Weber , A ndrew Reeves , W illiam H. Cernota , and Roy K. Wesley
18 E ngineering Gram-Negative Microbial Cell Factories
Using Transposon Vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Esteban Martínez-García , T omás Aparicio , V íctor de Lorenzo ,
and Pablo I. Nikel
19 P ERMutation Using Transposase Engineering (PERMUTE):
A Simple Approach for Constructing Circularly Permuted Protein Libraries . . . . . 2 95
Alicia M. Jones , J oshua T. Atkinson , and Jonathan J. Silberg
20 T ransposon Insertion Mutagenesis for Archaeal Gene Discovery . . . . . . . . . . . 3 09
Saija Kiljunen , M aria I. P ajunen , and Harri Savilahti
21 G enome-Wide Transposon Mutagenesis in M ycobacterium tuberculosis
and Mycobacterium smegmatis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Gaurav Majumdar , R endani Mbau , V inayak Singh , D igby F. Warner ,
Marte Singsås Dragset , and Raju Mukherjee
SECTION V SITE-DIRECTED MUTAGENESIS: PCR
AND DNA POLYMERASE-BASED METHODS
22 M ultiple Site-Directed and Saturation Mutagenesis
by the Patch Cloning Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Naohiro Taniguchi and Hiroshi Murakami
Contents ix
23 S eamless Ligation Cloning Extract (Slice) Method Using Cell Lysates
from Laboratory E scherichia coli Strains and its Application
to Slip Site-Directed Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 49
Ken Motohashi
24 F acile Site-Directed Mutagenesis of Large Constructs Using Gibson
Isothermal DNA Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 59
Isaac T. Yonemoto and Philip D. Weyman
25 R evised Mechanism and Improved Efficiency of the QuikChange
Site-Directed Mutagenesis Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Yongzhen Xia and Luying Xun
26 A n In Vitro Single-Primer Site-Directed Mutagenesis Method
for Use in Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 75
Yanchao Huang and Likui Zhang
27 U se of Megaprimer and Overlapping Extension PCR (OE-PCR)
to Mutagenize and Enhance Cyclodextrin Glucosyltransferase
(CGTase) Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Kian Mau Goh , K ok Jun Liew , K ian Piaw Chai , and Rosli Md Illias
SECTION VI IN VITRO MUTAGENESIS FOR STUDIES OF PROTEIN
STRUCTURE AND FUNCTION
28 S tep-By-Step In Vitro Mutagenesis: Lessons From Fucose-B inding
Lectin PA-IIL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Jana Mrázková , L enka Malinovská , and Michaela Wimmerová
29 A nalytical Methods for Assessing the Effects of Site-D irected
Mutagenesis on Protein–Cofactor and Protein–Protein Functional
Relationships. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 21
Esha Sehanobish , B rian A. Dow , and Victor L. Davidson
30 B iochemical and Biophysical Methods to Examine the Effects
of Site-Directed Mutagenesis on Enzymatic Activities
and Interprotein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 39
Misaki Kinoshita , J u Yaen Kim , Y uxi Lin , N atalia Markova ,
Toshiharu Hase , and Young-Ho Lee
31 U se of Random and Site-Directed Mutagenesis
to Probe Protein Structure–Function Relationships: Applied Techniques
in the Study of H elicobacter pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 61
Jeannette M. Whitmire and D. Scott M errell
SECTION VII RANDOM MUTAGENESIS: NOVEL PCR-BASED METHODS
32 N ovel Random Mutagenesis Method for Directed Evolution. . . . . . . . . . . . . . 4 83
Hong Feng , H ai-Yan Wang , and Hong-Yan Zhao
33 R andom Mutagenesis by Error-Prone Polymerase Chain Reaction
Using a Heavy Water Solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 91
Toshifumi Minamoto